Abstract: Myocardial infarction is a major cause of cardiac-related death in the US and those fortunate to survive the acute event suffer from chronic high risk of arrhythmia, stroke and congestive heart failure. Repairing the heart is difficult because cardiomyocytes are post-mitotic and cannot proliferate in order to regenerate damaged tissue. Recent work has demonstrated that cardiomyocytes can be derived from embryonic stem (ES) and induced pluripotent stem (iPS) cells, as well as transdifferentiated from other cells. However, organizing these cardiomyocytes to approximate or mimic the 3-D structure of stereotypical vascularized myocardium is still a major and unresolved challenge. Tissue engineering seeks to use polymeric scaffolds to organize cardiomyocytes and other cells such that cell- cell and cell-matrix interactions in concert with soluble factors guide tissue regeneration. While this has proven effective at short-term regeneration of cardiomyocyte-rich muscle, vascularization of these constructs and long- term survival have been quite limited and needs to be improved. Cardiogenesis during embryonic development produces vascularized myocardium, but current regenerative strategies do not mimic the extracellular matrix (ECM) that surrounds the cells in terms of composition or nanofibrillar architecture, nor are the mechanical properties matched to the myogenesis process. We hypothesize that the ECM, and especially fibronectin, in the developing heart encodes cues for the specification, differentiation and organization of cells into myocardium and that developmentally-staged, engineered matrices can recapitulate these cues in vitro, enabling a synthetic developmental pathway to tissue regeneration. To achieve this we are combining developmental biology, advanced 3-D imaging, materials science and cardiac tissue engineering. By combining such complementary and cutting-edge expertise, we are confident that our studies will yield new insights to actively move the field forward. To test our hypothesis, we will analyze the ECM in vivo during heart development and use the knowledge we gain to develop new 3-D cardiac tissue engineering techniques by: 1) Identifying the 3-D composition and structure of fibronectin in the vertebrate embryo during cardiac morphogenesis. We will use the chick embryo as an experimentally tractable model system. 2) Applying the ECM as the design template to engineer 3-D protein matrices that recapitulate the key compositional and structural features required for cardiac morphogenesis. 3) Engineering biomimetic, 3-D ECM nanofibrillar scaffolds and regenerate human myocardium using induced pluripotent stem cells. We will quantitatively assess the structure, molecular expression and electromechanical function relative to the native heart. Public Health Relevance: Myocardial infarction is a major cause of cardiac-related death in the US because the cardiomyocytes that survive are unable to repair the injured heart. Recent advances in stem cells have enabled derivation of new cardiomyocytes in large numbers, but organizing these cells into 3-D muscle tissue remains an unresolved challenge. We will determine how proteins in the extracellular matrix of the developing vertebrate heart guide cardiac morphogenesis and use that as the design template to engineer biomimetic protein scaffolds. This will enable us to tissue engineering 3-D human cardiac tissues, which will have future application in heart repair.